Engineered stimulus-responsive switches

Abstract

Ligand-responsive chimeric proteins are engineered to cause a detectable output in response to a preselected stimulus. The engineered chimeric proteins are useful in industrial, commercial, medical, and scientific fields as a tool for programming a cellular response to a stimulus of choice and for use with in vitro assays. The engineered chimeric proteins include a detection domain and an interaction domain. Interaction of the engineered chimeric protein with a target biomolecule is modulated by the presence or absence of the preselected stimulus.

1 - 66 . (canceled)  
     
     
         67 . A method of engineering a ligand-responsive chimeric protein construct that modulates gene expression responsive to the presence, concentration, or absence of a preselected ligand, the method comprising the steps of: 
 identifying one or more amino acid sequences that bind a preselected ligand using a recombinant display technique selected from the group consisting of phase display, retroviral display, bacterial surface display, yeast surface display, ribosome display, two-hybrid techniques, three-hybrid techniques, and derivatives thereof;    designing, based on the one or more amino acid sequences, an engineered peptide that binds the preselected ligand;    selecting an interaction domain which binds to a target biomolecule to modulate expression of a gene;    identifying a permissive position within or adjacent the interaction domain at which insertion of a heterologous peptide permits retention of binding of the interaction domain to the target biomolecule and conserves expression modulation activity; and    synthesizing a construct comprising the engineered peptide fused to the interaction domain at the permissive position,    thereby to produce a construct wherein binding of the ligand to the engineered peptide causes a change in said chimeric protein, the change regulating binding of the interaction domain to the target biomolecule and expression.    
     
     
         68 . (canceled)  
     
     
         69 . A method as in  claim 67 , wherein the engineered peptide is among the one or more amino acid sequences identified using the recombinant display technique.  
     
     
         70 . A method as in  claim 67 , wherein the engineered peptide reflects a consensus sequence derived from the one or more amino acid sequences identified.  
     
     
         71 . A method as in  claim 67 , wherein the engineered peptide is no more than one hundred amino acids in length.  
     
     
         72 . A method of engineering a stimulus-responsive chimeric protein construct which modulates expression of a preselected gene, the method comprising the steps of: 
 identifying a stimulus-responsive peptide of no more than one hundred amino acids in length;    selecting an interaction domain capable of binding to a target biomolecule to modulate transcription of a preselected gene;    identifying a permissive position within or adjacent the interaction domain at which insertion of a heterologous peptide permits binding of the interaction domain to the target biomolecule; and    synthesizing a construct comprising the stimulus-responsive peptide fused to the interaction domain at the permissive position,    thereby to produce a construct wherein recognition of the stimulus causes change in said chimeric protein, the change regulating binding of the interaction domain to the target biomolecule.    
     
     
         73 . A method as in  claim 71  or  72 , wherein the engineered peptide is no more than eighty amino acids in length.  
     
     
         74 . A method as in  claim 71  or  72 , wherein the engineered peptide is no more than sixty amino acids in length.  
     
     
         75 . A method as in  claim 71  or  72 , wherein the engineered peptide is no more than forty amino acids in length.  
     
     
         76 . A method as in  claim 71  or  72 , wherein the engineered peptide is no more than twenty amino acids in length.  
     
     
         77 . A method as in  claim 67  or  72 , wherein the permissive position is identified using stereochemical data about the three-dimensional structure of the interaction domain.  
     
     
         78 . A method as in  claim 67  or  72 , wherein the permissive position is identified using mutational data about the interaction domain.  
     
     
         79 . A method of engineering a stimulus-responsive chimeric protein construct, the method comprising the steps of: 
 identifying, from a database of information, a stimulus-responsive protein;    selecting an interaction domain capable of binding to a target biomolecule;    identifying a permissive position within or adjacent the interaction domain at which insertion of a heterologous peptide permits retention of binding of the interaction domain to the target biomolecule; and    synthesizing a construct comprising the stimulus-responsive protein, or a peptide derivative thereof, fused to the interaction domain at the permissive position,    thereby to produce a construct wherein receipt of the stimulus by the stimulus-responsive protein, or a peptide derivative thereof causes change in said chimeric protein, the change regulating binding of the interaction domain to the target biomolecule.    
     
     
         80 - 97 . (canceled)  
     
     
         98 . The construct produced by the method of  claim 67 .  
     
     
         99 . The construct produced by the method of  claim 72 .  
     
     
         100 . The construct produced by the method of  claim 79.